CN113336816B - Cytidine compounds and anti-tumor application thereof - Google Patents

Abstract

The invention relates to cytidine compounds and anti-tumor application thereof. In particular to a group of novel cytidine compounds with antitumor activity, a preparation method thereof and application in the antitumor aspect. The compound of the invention has a chemical structure shown in the following formula (I) and formula (II), wherein each substituent group is described in the specification. The compound prepared by the invention has excellent tumor inhibition effect on leukemia.

Description

Cytidine compounds and antitumor application thereof

Technical Field

The invention belongs to the field of medical chemistry, relates to a method for improving the antitumor performance of cytidine drugs, and particularly relates to a group of cytarabine prodrugs with excellent antitumor activity, a preparation method thereof, and applications thereof in the antitumor aspect.

Background

Cytarabine (Ara-C), also known as cytosine arabinoside, is chemically 1-beta-D-arabinofuranosyl-4-amino-2 (1H) -pyrimidinone having CAS number 147-94-4 and molecular formula C₉H₁₃N₃O₅Molecular weight 243.22, its chemical structure is shown in formula (VI) below:

cytarabine was first synthesized in 1959 by a pharmacologist such as Walwick R, at berkeley division, university of california. The FDA in the united states approved cytarabine for marketing in 6 months of 1969. The chemical structure of this drug, originally sold by Upjohn under the name Cytosar-U, is a nucleoside with cytosine bound to arabinose, hence the name "cytarabine".

Cytarabine is a pyrimidine antimetabolite which mainly acts on the proliferation stage of cells S and interferes with the proliferation of cells by inhibiting the synthesis of cell DNA. Cytarabine must be activated before entering human body to act, namely, cytarabine needs to be firstly converted into nucleotide 5-phosphate (Ara-CMP) under the catalysis of deoxycytidine kinase, Ara-CMp can react with proper nucleotide kinase to form nucleotide diphosphate and nucleotide triphosphate (Ara-CDP and Ara-CTP), the former can strongly inhibit the synthesis of DNA polymerase, and the latter can inhibit the conversion of cytidine diphosphate into deoxycytidine diphosphate, thereby inhibiting the polymerization and synthesis of cell DNA. The product is a cell cycle specific medicine, is most sensitive to the action of cells in S-phase proliferation stage, and has weak action on inhibiting RNA and protein synthesis.

Cytosine deaminase, which is widely present in solid tumors (but at low levels in leukemia cells), deaminates cytarabine to convert it to the inactive metabolite, uridine (Ara-U) (Shimma N, et al, Bioorganic & Medicinal Chemistry 8(2000)1697-1706), so this product (cytarabine hydrochloride by intravenous administration) is mainly used clinically in the induction remission stage or maintenance consolidation stage of acute lymphocytic and non-lymphocytic leukemia and in the acute phase of chronic myelocytic leukemia. Unfortunately, there are still many patients who cannot be relieved or relapse after relief, and the drug resistance factors account for a large proportion. The common adverse reactions of this herb include bone marrow depression such as leukopenia, thrombocytopenia and megaloblastic anemia, nausea and vomiting are also common. In addition, oral ulcers, thrombophlebitis and impaired liver function may occur. Although cytarabine is still one of the first-line drugs for clinically treating acute myeloid leukemia at present, the clinical application of the cytarabine is strictly limited due to the continuous aggravation of drug resistance and toxic and side effects.

The prior art still expects a new method and expects to have some or some more excellent effect on treating tumors, for example, the antitumor drug cytosine compound such as cytosine arabinoside prodrug and the composition thereof with more excellent performance are expected to be applied to clinic.

Disclosure of Invention

The object of the present invention is to provide a novel method which is expected to have some or some more excellent effects for treating tumors, for example, to provide an antitumor agent having more excellent properties for clinical use. The present inventors have surprisingly found that compounds having the structure of the present invention exhibit one or more excellent effects as antitumor agents. The present invention has been completed based on such findings.

To this end, the present invention provides, in a first aspect, compounds of the following formula (I) (which may also be referred to herein as cytarabine carbamate compounds or cytarabine carbamate) and compounds of the formula (II) (which may also be referred to herein as cytarabine triester compounds),

or a pharmaceutically acceptable salt, solvate or solvate thereof,

wherein:

R₁、R₂、R₃independently represent C_6-22Straight or branched (e.g. C)_8-22Straight or branched, e.g. C_10-22Linear or branched) saturated alkyl or unsaturated alkenyl, in which 1 or 2 CH groups in the carbon chain are present₂Optionally substituted by O;

x represents O, NH or is absent.

A compound according to the first aspect of the invention, wherein R is₁、R₂、R₃Selected from: n-decyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, n-docosyl, n-hexadecyloxypropyl, n-octadecyl, n-9-ene) n-decyl, (11-ene) n-dodecyl, (11-ene) n-dodecylethyl.

A compound according to the first aspect of the invention or any other aspect of the invention, wherein the pharmaceutically acceptable salt is a salt with an inorganic acid or with an organic acid.

A compound according to the first aspect of the invention or as referred to in any of the other aspects of the invention, wherein the inorganic acid is selected from: hydrochloric acid, sulfuric acid, phosphoric acid. A particularly preferred pharmaceutically acceptable salt is the hydrochloride salt.

A compound according to the first aspect of the invention or as referred to in any of the other aspects of the invention, wherein the organic acid is selected from: acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, malic acid, amino acids (e.g. alanine, aspartic acid, lysine), sulfonic acids (e.g. methanesulfonic acid, p-toluenesulfonic acid).

The compounds according to the first aspect of the invention may also exist in the form of solvates (e.g. hydrates), and therefore such solvates (e.g. hydrates) are also included in the compounds of the invention.

A compound according to the first aspect of the invention, which is compound 1 to compound 17 selected from:

compound 1: n is a radical of⁴-n-tetradecyloxycarbonyl cytarabine,

compound 2: n is a radical of⁴-n-hexyloxycarbonyl cytarabine,

compound 3: n is a radical of⁴-n-decyloxycarbonyl cytarabine,

compound 4: n is a radical of⁴-n-eicosyloxycarbonyl cytarabine,

compound 5: n is a radical of⁴-n-docosanyloxycarbonyl cytarabine,

compound 6: n is a radical of⁴- (9-decenyloxycarbonyl) cytarabine,

compound 7: n is a radical of⁴- (13-tetradecyloxycarbonyl) cytarabine,

compound 8: n is a radical of⁴- (21-docosadienyloxycarbonyl) cytarabine,

compound 9: 2 ', 3 ', 5 ' -tri-O-n-tetradecanoyl cytarabine,

compound 10: 2 ', 3 ', 5 ' -tri-O-n-undecylcytarabine,

compound 11: 2 ', 3', 5 '-tri-O- (21' -n-docosadienoyl) cytarabine,

compound 12: 2 ', 3 ', 5 ' -tri-O- (n-hexyloxyacyl) cytarabine,

compound 13: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoyloxyacyl) cytarabine,

compound 14: 2 ', 3', 5 '-tri-O- (21' -n-docosadienyloxyformyl) cytarabine,

compound 15: 2 ', 3 ', 5 ' -tri-O- (n-decylaminoyl) cytarabine,

compound 16: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoylamino acyl) cytarabine,

compound 17: 2 ', 3 ', 5 ' -tri-O- (21 "-n-eicosadienoyl) cytarabine.

Further, the second aspect of the present invention provides a process for preparing a compound of formula (I) and a compound of formula (ii), which are carried out according to the following reaction scheme one and reaction scheme two, respectively:

the first reaction route is as follows:

in scheme one, R₁As defined in any one of the embodiments of the first aspect of the invention.

Reaction scheme two:

in scheme two, R₁、R₂、R₃And X is as defined in any one of the embodiments of the first aspect of the invention.

A process according to the second aspect of the invention, wherein the preparation of the compound of formula (I) comprises the steps of:

1) dissolving the compound of formula (III) in a non-polar solvent (such as dichloromethane, chloroform, tetrahydrofuran, dioxane), and reacting with 1-2 times of the equivalent of the compound of formula (IV) in the presence of 1.5-3 times of the equivalent of an organic base (such as triethylamine, pyridine, 4-dimethylaminopyridine) at 0-40 ℃ for 3-10 hours with stirring to obtain the compound of formula (V);

2) dissolving the compound of formula (V) in a protic solvent (e.g., water, alcohol or an alcohol-water mixed solvent), adding 0.1 to 2 equivalents of an inorganic base (e.g., 0.1 to 0.5 equivalent; e.g., sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate), at 0 deg.C to room temperature for 0.5 to 3 hours with stirring to provide the compound of formula (I).

The process according to the second aspect of the present invention, wherein the preparation of the compound of formula (II) comprises the steps of:

1) dissolving a compound of formula (VI) in a non-polar solvent selected from: dichloromethane, trichloromethane, tetrahydrofuran, dioxane, in the presence of an organic base selected from the group consisting of: triethylamine, pyridine, 4-dimethylaminopyridine and 1-6 times of equivalent weight of Boc₂Stirring and reacting O at room temperature to 50 ℃ for 3-15 hours to obtain an amino-protected compound of formula (VII);

2) dissolving a compound of formula (VII) in a non-polar solvent selected from: dichloromethane, trichloromethane, tetrahydrofuran, dioxane, in the presence of an organic base selected from the group consisting of: triethylamine, pyridine and 4-dimethylamino pyridine react with 1-4 times of equivalent of a compound of formula (IV), a compound of formula (VIII) or a compound of formula (IX) for 4-15 hours under stirring at 0-room temperature to obtain a compound of formula (X);

3) dissolving a compound of formula (X) in a non-polar solvent selected from: adding 1-2 times of equivalent of acid selected from the following into dichloromethane, trichloromethane, tetrahydrofuran and dioxane: and (3) stirring trifluoroacetic acid and hydrochloric acid at 0-room temperature for reaction for 0.5-5 hours to obtain the compound of the formula (II).

Further, in a third aspect, the present invention provides the use of a compound according to any one of the first aspect of the present invention or a compound prepared by the process according to any one of the second aspect of the present invention in the manufacture of a medicament for the treatment of a tumour or ophthalmia in a mammal.

The use according to the third aspect of the invention, wherein the tumour is selected from the group consisting of: leukemia (e.g., acute leukemia such as acute myelocytic leukemia, acute monocytic leukemia, acute lymphocytic leukemia), malignant lymphoma, lung cancer, cancer of the digestive tract, and head and neck cancer.

The use according to the third aspect of the invention, wherein the ophthalmia is selected from: viral keratitis, epidemic conjunctivitis.

The use according to the third aspect of the invention, wherein the compound elicits less adverse effects of thrombocytopenia in a mammal than cytarabine, e.g. a statistically significant difference with a p-value < 0.05.

Further, in a fourth aspect of the present invention, there is provided a method for reducing adverse effects of cytarabine on thrombocytopenia in a mammal, which comprises the step of preparing a compound of formula (I) or a compound of formula (II) as described in any one of the first aspect of the present invention from cytarabine. In one embodiment of the invention, the mammal is a human.

Any technical feature possessed by any one aspect of the invention or any embodiment of that aspect is equally applicable to any other embodiment or any embodiment of any other aspect, so long as they are not mutually inconsistent, although appropriate modifications to the respective features may be made as necessary when applicable to each other. Various aspects and features of the disclosure are described further below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention has been described generally and/or specifically with respect to materials used in testing and testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible.

Examples of preparation of Compounds

Example 1、N⁴-n-tetradecyloxycarbonyl cytarabine

2 ', 3 ', 5 ' -tri-O-benzoylCytarabine (III, 555mg,1.0mmol) was dissolved in dichloromethane (15ml), and triethylamine (417. mu.l, 3.0mmol) and tetradecyl chloroformate (IV) were added in that order^/333mg,1.2 mmol). Stirring for 8-15 hours at room temperature, and finishing the reaction. And (3) post-treatment: the reaction system was washed with water, dried over anhydrous sodium sulfate, filtered and concentrated, and the residue was used directly in the next reaction. 2 ', 3 ', 5 ' -tri-O-benzoyl cytarabine can be readily prepared by methods well known in the art, for example, from cytarabine as a starting material.

The product V obtained in the previous step^/Dissolved in methanol (10mL), and a sodium hydroxide solution (0.5mL,1M) was added thereto, followed by stirring at room temperature for 1 hour to complete the reaction. And (3) post-treatment: the reaction solution was concentrated, and the residue was separated and purified by column chromatography (dichloromethane: methanol 25: 1 to 10: 1) to give a pure white solid (I)^/) For the title compound, HPLC purity>98.6 percent. The content or purity of the target substance in various materials (including raw material medicines and compositions) is determined by an HPLC method under the following conditions: inertsil ODS-SP column (5 μm, 150 mm. times.4.6 mm), purified with methanol: water (80:20) is used as a mobile phase for isocratic elution, the flow rate is 1.0mL/min, the detection wavelength is 241nm, the column temperature is 30 ℃, and the sample injection amount is 20 mu L.

¹H NMR(500MHz,DMSO)δ9.08(s,1H),8.03(d,J＝7.5Hz,1H),7.01(d,J＝7.6Hz,1H),6.04(d,J＝3.9Hz,1H),5.48(d,J＝5.3Hz,2H),5.07(d,J＝5.1Hz,1H),4.09(t,J＝6.6Hz,2H),4.04(d,J＝5.9Hz,1H),3.92(d,J＝3.4Hz,1H),3.82(d,J＝3.6Hz,1H),3.61(t,J＝5.6Hz,2H),1.59(t,J＝7.1Hz,2H),1.24(d,J＝6.8Hz,22H),0.85(t,J＝6.8Hz,3H).MS-ESI(m/z):483.3(M+H)⁺.

Example 2、N⁴-n-hexyloxycarbonyl cytarabine

Referring to example 1, compound III was substituted with n-hexyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 98.3%.

¹H NMR(500MHz,DMSO)δ9.13(s,1H),7.77(d,J＝7.7Hz,1H),6.91(d,J＝7.7Hz,1H),5.98(d,J＝6.0Hz,1H),5.16(d,J＝4.4Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.9,3.1Hz,1H),3.72–3.60(m,2H),1.72–1.63(m,1H),1.43(dp,J＝8.1,6.9Hz,2H),1.38–1.24(m,6H),0.89(t,J＝6.8Hz,3H).MS-ESI(m/z):371.2(M+H)⁺.

Example 3、N⁴-n-decyloxycarbonyl cytarabine

Referring to example 1, substitution of compound III with n-decyl chloroformate gave, after removal of the Bz-protecting group, the title compound as a white solid with HPLC purity > 98.7%.

¹H NMR(500MHz,DMSO)δ9.11(s,1H),7.90(d,J＝7.7Hz,1H),6.78(d,J＝7.7Hz,1H),6.19(d,J＝6.0Hz,1H),5.03(d,J＝4.4Hz,1H),4.84(d,J＝4.7Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.72–3.60(m,2H),1.71–1.62(m,2H),1.44–1.34(m,2H),1.34–1.22(m,6H),1.26(s,6H),0.95–0.84(m,3H).MS-ESI(m/z):427.2(M+H)⁺.

Example 4、N⁴-n-eicosyloxycarbonyl cytarabine

Referring to example 1, compound iii was substituted with n-eicosyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 98.1%.

¹H NMR(500MHz,DMSO)δ7.90(d,J＝7.7Hz,1H),6.71(d,J＝7.7Hz,1H),6.21(d,J＝6.0Hz,1H),5.19(d,J＝4.4Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.02(td,J＝6.8,4.5Hz,1H),3.89(dt,J＝6.8,3.0Hz,1H),3.72–3.60(m,2H),1.67(tt,J＝7.7,6.4Hz,2H),1.44(dt,J＝8.1,7.0Hz,2H),1.37–1.25(m,32H),0.93–0.85(m,3H).MS-ESI(m/z):567.4(M+H)⁺.

Example 5、N⁴-n-docosanyloxycarbonyl cytarabine

Referring to example 1, compound iii was substituted with n-docosyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 97.7%.

¹H NMR(500MHz,DMSO)δ8.88(s,1H),7.94(d,J＝7.7Hz,1H),6.68(d,J＝7.7Hz,1H),6.14(d,J＝6.0Hz,1H),5.03(d,J＝4.4Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.01(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.72–3.60(m,2H),1.67(tt,J＝7.7,6.4Hz,2H),1.44–1.34(m,2H),1.33–1.22(m,36H),0.93–0.85(m,3H).MS-ESI(m/z):595.4(M+H)⁺.

Example 6、N⁴- (9-decenyloxycarbonyl) cytarabine

Referring to example 1, compound III was substituted with 9-decenyl chloroformate and the Bz protecting group removed to give the title compound as a white solid with HPLC purity > 98.3%.

¹H NMR(500MHz,DMSO)δ8.96(s,1H),7.89(d,J＝7.7Hz,1H),6.62(d,J＝7.7Hz,1H),6.12(d,J＝6.0Hz,1H),5.80(tt,J＝10.2,6.8Hz,1H),5.08(ddt,J＝10.3,2.1,1.0Hz,1H),5.00(d,J＝4.4Hz,1H),4.97(ddt,J＝10.3,2.1,0.9Hz,1H),4.84(d,J＝4.8Hz,1H),4.69(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.66(ddd,J＝4.4,3.1,1.1Hz,2H),2.23(tdt,J＝7.8,6.7,1.0Hz,2H),1.77–1.65(m,1H),1.39(dq,J＝8.1,6.7Hz,2H),1.34–1.22(m,8H).MS-ESI(m/z):425.2(M+H)⁺.

Example 7、N⁴- (13-tetradecenyloxycarbonyl) cytarabine

Referring to example 1, compound III was substituted with (13-tetradecenyl) chloroformate and the Bz-protecting group was removed to give the title compound as a white solid with HPLC purity > 98.6%.

¹H NMR(500MHz,DMSO)δ8.77(s,1H),7.90(d,J＝7.7Hz,1H),6.79(d,J＝7.7Hz,1H),6.03(d,J＝6.0Hz,1H),5.80(tt,J＝10.2,6.8Hz,1H),5.28(ddt,J＝10.3,2.1,1.0Hz,1H),5.00(d,J＝4.4Hz,1H),4.97(ddt,J＝10.2,2.2,0.9Hz,1H),4.84(d,J＝4.8Hz,1H),4.73(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.11(td,J＝6.8,4.5Hz,1H),3.93(dt,J＝6.8,3.0Hz,1H),3.58(ddd,J＝4.4,3.1,1.1Hz,2H),2.03(tdt,J＝7.8,6.7,1.0Hz,2H),1.71–1.62(m,2H),1.39(dq,J＝8.1,7.1Hz,2H),1.34–1.22(m,16H).MS-ESI(m/z):481.3(M+H)⁺.

Example 8、N⁴- (21-docosadienyloxycarbonyl) cytarabine

Referring to example 1, substitution of compound III with 21-dodecenyl chloroformate (21-docodecenyl ester) gave the title compound as a white solid after removal of the Bz-protecting group, with an HPLC purity of > 97.3%.

¹H NMR(500MHz,DMSO)δ9.04(s,1H),7.33(d,J＝7.7Hz,1H),6.82(d,J＝7.7Hz,1H),6.00(d,J＝6.0Hz,1H),5.79(tt,J＝10.2,6.8Hz,1H),5.08(ddt,J＝10.3,2.1,1.0Hz,1H),5.00(d,J＝4.4Hz,1H),4.97(ddt,J＝10.3,2.2,1.0Hz,1H),4.84(d,J＝4.8Hz,1H),4.79(t,J＝4.5Hz,1H),4.24(ddd,J＝6.9,5.9,4.7Hz,1H),4.15(t,J＝6.4Hz,2H),4.08(td,J＝6.8,4.5Hz,1H),3.86(dt,J＝6.8,3.0Hz,1H),3.66(ddd,J＝4.3,3.1,1.1Hz,2H),2.08–1.99(m,2H),1.67(tt,J＝7.7,6.4Hz,2H),1.44–1.34(m,2H),1.34–1.22(m,32H).MS-ESI(m/z):593.4(M+H)⁺.

Example 92 ', 3 ', 5 ' -tri-O-n-tetradecanoyl cytarabine

Cytarabine (VI, 244mg,1.0mmol) was dissolved in N, N-dimethylformamide (15ml), and Boc was added at 0 deg.C₂O (437mg,2.0mmol) was added to the solution, stirred for 10 minutes, heated to 60 ℃ and stirred for 6-8 hours. And (3) post-treatment: the reaction solution was washed with water, and extracted with ethyl acetate (10 ml). The combined extracts were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Separating and purifying the residue by column chromatography to obtain a compound VII^/. Compound VII^/Dissolved in a dichloromethane solution (10ml), triethylamine (417. mu.l, 3mmol) and tetradecanoyl chloride (VIII) were added successively^/890mg, 3.6mmol) to the reaction solution, and after stirring at room temperature for 3 to 6 hours, the reaction was completed. And (3) post-treatment: the reaction solution was concentrated to obtain a residue (X)^/) Directly used for the next reaction.

Product X described above^/Dissolving in dichloromethane solution (10ml), adding trifluoroacetic acid (2ml) to remove protecting group to obtain white solid compound II^/For titlingCompound, HPLC purity>97.6％。

¹H NMR(500MHz,CDCl₃)δ7.58(t,J＝5.6Hz,1H),6.33(d,J＝3.9Hz,1H),5.71(dd,J＝7.6,4.6Hz,1H),5.48(d,J＝3.8Hz,1H),5.01(s,1H),4.44(dd,J＝11.8,7.2Hz,1H),4.34(dd,J＝11.8,4.0Hz,1H),4.15(dt,J＝7.1,3.6Hz,1H),2.40–2.31(m,4H),2.27–2.14(m,2H),1.63(q,J＝6.9Hz,4H),1.54–1.46(m,2H),1.36–1.18(m,61H),0.87(t,J＝6.8Hz,9H).MS-ESI(m/z):915.7(M+H)⁺.

Example 102 ', 3 ', 5 ' -tri-O-n-undecylcytarabine

Referring to example 9, the substitution reaction of compound VII with n-undecanoyl chloride, deprotection of the Bz protecting group gave the title compound as a white solid with HPLC purity > 98.1%.

¹H NMR(500MHz,CDCl₃)δ7.88(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.17(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.43(dd,J＝6.8,4.2Hz,1H),5.25–5.21(m,1H),4.33(dt,J＝6.3,3.9Hz,1H),4.23(dd,J＝11.9,4.0Hz,1H),4.15(dd,J＝11.8,3.9Hz,1H),2.35–2.26(m,6H),1.60–1.49(m,6H),1.34–1.19(m,42H),0.91–0.85(m,9H).MS-ESI(m/z):747.5(M+H)⁺.

Example 112 ', 3', 5 '-tri-O- (21' -n-docosadienoyl) cytarabine

Referring to example 9, the compound VII was substituted with 21 "-n-docosenylformyl chloride to remove the Bz protecting group to give the title compound as a white solid with HPLC purity > 98.5%.

¹H NMR(500MHz,CDCl₃)δ7.86(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.17(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.80(tt,J＝10.0,6.9Hz,3H),5.43(dd,J＝6.8,4.2Hz,1H),5.26–5.21(m,1H),5.12–5.04(m,3H),5.01–4.93(m,3H),4.33(dt,J＝6.4,4.0Hz,1H),4.19(d,J＝3.9Hz,2H),2.35–2.26(m,6H),2.08–1.99(m,6H),1.56(dddd,J＝34.0,16.4,8.6,7.3Hz,6H),1.27(ddd,J＝5.8,3.4,2.0Hz,102H).MS-ESI(m/z):1246.1(M+H)⁺.

Examples122 ', 3 ', 5 ' -tri-O- (n-hexyloxyacyl) cytarabine

Referring to example 9, substitution of compound VII with n-hexyl chloroformate and removal of Bz protecting group gave the title compound as a white solid with HPLC purity > 97.3%.

¹H NMR(500MHz,CDCl₃)δ7.72(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.18(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.47(dd,J＝6.7,4.1Hz,1H),5.37(t,J＝6.5Hz,1H),4.79(dt,J＝6.1,4.0Hz,1H),4.38(dd,J＝3.9,2.1Hz,2H),4.22–4.13(m,6H),1.71(tt,J＝7.3,6.2Hz,6H),1.44–1.24(m,18H),0.89(t,J＝6.7Hz,9H).MS-ESI(m/z):627.3(M+H)⁺.

Example 132 ', 3 ', 5 ' -tri-O- (n-tetradecanoyloxy) cytarabine

Referring to example 9, the substitution reaction of compound VII with n-tetradecyl chloroformate and removal of Bz protecting group gave the title compound as a white solid with HPLC purity > 98.6%.

¹H NMR(500MHz,CDCl₃)δ7.80(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.18(d,J＝4.1Hz,1H),6.08(d,J＝7.3Hz,1H),5.47(dd,J＝6.7,4.1Hz,1H),5.37(t,J＝6.5Hz,1H),4.79(dt,J＝6.1,4.0Hz,1H),4.42–4.34(m,2H),4.17(td,J＝6.3,1.8Hz,6H),1.72(tt,J＝7.7,6.2Hz,6H),1.39(p,J＝7.3Hz,6H),1.34–1.21(m,60H),0.93–0.85(m,9H).MS-ESI(m/z):963.7(M+H)⁺.

Example 142 ', 3', 5 '-tri-O- (21' -n-docosadienyloxyformyl) cytarabine

Referring to example 9, substitution of compound VII with chloroformic acid (21-docodecenyl ester) and removal of the Bz protecting group gave the title compound as a white solid with HPLC purity > 98.4%.

¹H NMR(500MHz,CDCl₃)δ7.76(d,J＝7.3Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.18(d,J＝4.2Hz,1H),6.08(d,J＝7.3Hz,1H),5.80(tt,J＝10.3,6.9Hz,3H),5.47(dd,J＝6.7,4.1Hz,1H),5.42–5.32(m,1H),5.12–5.04(m,3H),5.01–4.93(m,3H),4.79(dt,J＝6.1,3.9Hz,1H),4.43–4.34(m,2H),4.17(td,J＝6.1,1.7Hz,6H),2.03(dtd,J＝8.0,6.8,1.1Hz,6H),1.72(tt,J＝7.6,6.2Hz,6H),1.39(p,J＝7.3Hz,6H),1.38–1.21(m,96H).MS-ESI(m/z):1294.0(M+H)⁺.

Example 152 ', 3 ', 5 ' -tri-O- (n-decylaminoyl) cytarabine

Referring to example 9, the substitution reaction of compound VII with n-decyl isocyanate, deprotection of the Bz protecting group gave the title compound as a white solid with HPLC purity > 98.2%.

¹H NMR(500MHz,CDCl₃)δ7.68(d,J＝7.5Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.14(d,J＝3.8Hz,1H),6.08(d,J＝7.3Hz,1H),5.41(dd,J＝6.8,3.8Hz,1H),5.27(dd,J＝6.8,5.9Hz,1H),5.16–5.06(m,2H),4.39–4.30(m,2H),3.15(q,J＝5.5Hz,2H),3.03(dq,J＝13.9,5.5Hz,1H),2.90–2.79(m,2H),1.53–1.41(m,6H),1.33–1.24(m,42H),0.93–0.85(m,9H).MS-ESI(m/z):792.6(M+H)⁺.

Example 162 ', 3 ', 5 ' -tri-O- (n-tetradecanoylamino acyl) cytarabine

Referring to example 9, the substitution reaction of compound VII with n-tetradecyl isocyanate followed by the removal of the Bz protecting group gave the title compound as a white solid with HPLC purity > 97.9%.

¹H NMR(500MHz,CDCl₃)δ7.85(d,J＝7.5Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.14(d,J＝3.8Hz,1H),6.08(d,J＝7.3Hz,1H),5.43–5.40(m,1H),5.27(dd,J＝6.8,5.9Hz,1H),5.16–5.06(m,2H),4.36(ddd,J＝5.7,4.5,3.5Hz,1H),4.33–4.31(m,2H),3.15(q,J＝5.5Hz,2H),3.03(dq,J＝13.9,5.5Hz,1H),2.89–2.81(m,2H),1.53–1.41(m,6H),1.34–1.23(m,66H),0.93–0.85(m,9H).MS-ESI(m/z):960.8(M+H)⁺.

Example 172 ', 3', 5 '-tri-O- (21' -n-eicosadienoyl) cytarabine

Referring to example 9, substitution of compound VII with (21-docodecenyl isocyanate) and removal of the Bz protecting group gave the title compound as a white solid with HPLC purity > 98.4%.

¹H NMR(500MHz,CDCl₃)δ7.77(d,J＝7.5Hz,1H),6.91(d,J＝6.0Hz,1H),6.81(d,J＝6.0Hz,1H),6.14(d,J＝3.8Hz,1H),6.08(d,J＝7.3Hz,1H),5.80(tt,J＝10.3,6.9Hz,3H),5.41(dd,J＝6.8,3.8Hz,1H),5.28–5.25(m,1H),5.16–5.04(m,6H),4.97(dt,J＝13.7,3.1Hz,3H),4.39–4.28(m,3H),3.15(q,J＝5.5Hz,2H),3.03(dq,J＝13.9,5.5Hz,1H),2.90–2.80(m,2H),2.03(td,J＝7.9,6.8Hz,6H),1.53–1.41(m,6H),1.35–1.23(m,102H).MS-ESI(m/z):1291.1(M+H)⁺.

The yields in the steps from compound III to compound V in examples 1 to 8 above are 57 to 64% (e.g., 61.6% in example 1), and the yields in the steps from compound VII to compound X in examples 9 to 17 are 52 to 58% (e.g., 56.3% in example 9). In a supplementary example of the present invention (which may be referred to as example 18 in the present invention), with reference to examples 1 to 8, respectively, except that sodium pyruvate was further added together with compound III in the step from compound III to compound V to obtain the title compound; with reference to examples 9 to 17, respectively, except that 0.1 molar equivalent of sodium pyruvate with respect to compound VII was further added along with compound VII in the step from compound VII to compound X to obtain the title compound; it was found that the yield of the step from compound III to compound V in this additional example was 77 to 81% (e.g., 78.4% in the additional example of reference example 1), and the yield of the step from compound VII to compound X in this additional example was 71 to 77% (e.g., 74.7% in the additional example of reference example 9). It has surprisingly been found that in the process according to the invention for preparing the compounds, the addition of 0.1 molar equivalents of sodium pyruvate together with compound III in the step from compound III to compound V or 0.1 molar equivalents of sodium pyruvate together with compound VII in the step from compound VII to compound X significantly increases the reaction yield. Thus, according to any aspect of the present invention, wherein in the preparation of the compound of formula (I) or the compound of formula (II), 0.1 molar equivalent of sodium pyruvate is further added with compound III in the preparation step from compound III to compound V, and 0.1 molar equivalent of sodium pyruvate is further added with compound VII in the preparation step from compound VII to compound X.

Second, biological test example section of Compounds

Test example 1 Effect of Compounds on platelets

Clinical use of cytarabine is known to cause thrombocytopenic adverse reactions, which in turn affects the blood coagulation function. This experiment examined the in vivo performance of the drug by measuring the effect on the platelets and clotting time of animals after administration of the compounds of the invention. All data are expressed as mean ± SD, and comparisons between groups were performed using the t-test.

1. Material

White Kunming mice, each half of male and female, with the weight of 18-22 g, are purchased from the breeding house; cytarabine hydrochloride for injection (H20084073); the sterilized aqueous solution of the products of the embodiments 1-17 of the invention is prepared on site; inverted microscope XDS-900 (Zeikang).

2. Grouping

Test mice were taken and randomly grouped into 10 groups, each group including: a normal control group, b cytarabine group, c compound group (including c1 group using the compound of example 1, c2 group using the compound of example 2, and c17 group using the compound of example 17 for ….

3. Composition medicine

a normal control group is injected with 0.9 percent sodium chloride injection with the same volume as that of the cytarabine group b by intraperitoneal injection every day; b, the cytarabine group is administrated with 0.1mmol/kg/d of cytarabine by intraperitoneal injection every day; c, the corresponding compound is administered by intraperitoneal injection every day at a rate of 0.1 mmol/kg/d; each group of medicines is prepared into solution by using 0.9 percent sodium chloride injection as a solvent according to the concentration of the liquid medicine volume of 0.1 to 0.2ml per animal for intraperitoneal injection. The above dose was administered daily for 7 days, and the general condition, activity, infection, bleeding, food intake, etc. of the animals were observed daily.

4. The reference (Chenqi, methodology of pharmacological research in Chinese medicine, Beijing: people's health Press, 1994: 484-: blood was drawn from the orbital capillary, dropped onto a slide glass, and the drop diameter was about 5mm, immediately timed with a stopwatch, the day before the administration (0d) and 6h after the last administration (7d), respectively. The clean No. 4 injection needle is slightly shifted inwards from the edge of the blood drop to observe whether blood filaments are picked up or not, and the blood coagulation time is determined from the beginning of blood sampling to the end of the blood filaments picking up.

As a result: the pre-dose clotting time(s) was summarized in data for all groups of animals as 118.24 ± 26.47; after 7 days of administration, the coagulation time(s) was 282.24 ± 43.72 ×, that of the b cytarabine group was 176.63 ± 38.46 × # in the C1 group, and the mean values of the coagulation time(s) of the C2 to C17 groups were 161 to 195, significantly different from 0 day by p <0.01, and significantly different from 7 days of the b cytarabine group by # p < 0.01. Wherein # is p <0.01 compared to day 0 results and # is p <0.01 compared to day 7 b cytarabine group results.

5. Reference (national institute of health, national operating rules for clinical laboratory, Nanjing: university Press 1997: 22-23) method for blood cell counts: blood was collected one day before administration (day 0) and one day after administration (day 8), and peripheral blood platelets (Bpc) were counted manually using a microscope, according to "visual counting" of "platelet count" of "national clinical laboratory practice". Results are expressed as Bpc change rates: bpc change rate (0 days Bpc mean-8 days Bpc mean)/0 days Bpc mean × 100%.

As a result: bpc numbers (x 1000/. mu.L) were collected at day 0 before dosing to 847.16. + -. 138.34 for all groups of animals; 7 days after administration, the number of Bpc in the b cytarabine group (x 1000/. mu.L) was 402.73 ± 86.54 #, the number of Bpc in the C1 group (x 1000/. mu.L) was 683.48 ± 116.58 #, and the mean values of Bpc in the C2-C17 groups (x 1000/. mu.L) were ##orwithin a range of 653-698, wherein p is <0.01 compared with 0 day results, p is <0.05 compared with 0 day results, and # is p <0.01 compared with 7 day b cytarabine group results; the change rate of Bpc on day 8 in each group was 52.46% for cytarabine, 19.32% for C1, and 17.6-22.9% for C2-C17.

According to the results of this test, it was unexpectedly found that the compound of the present invention is significantly weaker than cytarabine in causing platelet lowering at the equivalent dose, and thus it can be expected that the compound of the present invention will cause significantly less adverse effects in causing platelet lowering. Although the test examples were conducted using mice, it is obvious to those skilled in the art that the results of such animal models can be extrapolated to the results of other mammals such as humans.

Test example 2 pharmacodynamic study of Compound

The influence of the compound on the cell biological characteristics of the acute myeloid leukemia SCID model mouse is examined by referring to the method of Corhong literature (Corhong, et al, Epimedium extract on the cell biological characteristics of the acute myeloid leukemia SCID model mouse, Chinese medicine emergency 2018 (03): 389-393). The HL-60 cell line, namely, the human promyelocytic leukemia cell is obtained from a 36-year-old female suffering from acute promyelocytic leukemia at the national cancer institute, mainly a neutrophil promyelocytic cell, and is a cell line used for laboratory research on how certain blood cells are formed, particularly for the research on the anti-tumor activity of acute myeloid leukemia drugs. HL-60 cells were purchased from Punuisal, Inc., and the remaining materials were commercially available.

Cell culture was performed according to the method of the Kyoho literature. The test mice were randomly grouped into 10 mice each, including: a normal control group, b cytarabine group, c compound group (including c1 group using the compound of example 1, c2 group using the compound of example 2, and c17 group using the compound of example 17 for …), and d model group (no administration was performed after successful molding). And (4) molding by referring to the method of the Corohong literature and reaching the standard of nodulation, wherein the normal control group is not molded, and the other groups are molded.

Administration: after the injection of HL-60 cell suspension, at the last 1d of the 3 rd week, a normal control group and a model group d, 0.9 percent sodium chloride injection with the same volume as that of the cytarabine group b is given to the two groups of the normal control group a and the model group d by intraperitoneal injection every day; b Cytarabine group is administered with 0.4mmol/m Cytarabine per day by intraperitoneal injection²D; c group was administered the corresponding compound 0.4mmol/m by intraperitoneal injection every day²D; each group of medicines is prepared into a solution by using 0.9 percent sodium chloride injection as a solvent according to the concentration that the volume of a liquid medicine injected into the abdominal cavity every time is 0.1 to 0.2ml per animal. Each timeThe above dose was administered daily for 7 days, and the general condition, activity, infection, bleeding, food intake, etc. of the animals were observed daily.

Collecting and detecting the sample by referring to the method in the Ki hong literature, taking tail vein blood of the animal 48 hours after the last administration, and counting the number (%) of HL-60 cells by using an artificial counting method, and obtaining the results: the cell numbers (%) of HL-60 in the a normal control group and HL-60 in the b cytarabine group were 6.62 +/-0.87, the cell numbers (%) of HL-60 in the C1 group were 5.12 +/-0.63, the average values of the cell numbers (%) of HL-60 in the C2-C17 groups were 4.3-6.4, and the cell numbers (%) of HL-60 in the d model group were 19.73 +/-2.04, wherein p is less than 0.05 compared with the results in the d model group.

The above results indicate that the compounds of the present invention are slightly superior to cytarabine in inhibiting HL-60 cells but with no statistically significant difference.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein.

Claims (6)

1. A compound of formula (II):

or a pharmaceutically acceptable salt thereof, wherein:

R₁、R₂、R₃each independently represents C_6-22Saturated alkyl or unsaturated alkenyl, straight or branched, in which 1 CH of the carbon chain is₂Optionally substituted with O;

x represents O or NH.

2. According to claim 1A compound wherein said R₁、R₂、R₃Selected from: n-decyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-hexadecyl, n-octadecyl, n-eicosyl, n-docosyl, n-hexadecyloxypropyl, n-octadecyl, n-9-ene) n-decyl, (11-ene) n-dodecyl, (11-ene) n-dodecylethyl.

3. The compound according to claim 1, said pharmaceutically acceptable salt being a salt with an inorganic or organic acid selected from the group consisting of: hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, trifluoroacetic acid, citric acid, maleic acid, oxalic acid, succinic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, ascorbic acid, malic acid, alanine, aspartic acid, lysine, methanesulfonic acid, p-toluenesulfonic acid.

4. The compound according to claim 1, which is compound 12 to compound 17 selected from the group consisting of:

compound 12: 2 ', 3 ', 5 ' -tri-O- (n-hexyloxyacyl) cytarabine,

compound 13: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoyloxyacyl) cytarabine,

compound 14: 2 ', 3', 5 '-tri-O- (21' -n-docosadienyloxyformyl) cytarabine,

compound 15: 2 ', 3 ', 5 ' -tri-O- (n-decylaminoyl) cytarabine,

compound 16: 2 ', 3 ', 5 ' -tri-O- (n-tetradecanoylamino acyl) cytarabine,

compound 17: 2 ', 3 ', 5 ' -tri-O- (21 "-n-eicosadienoyl) cytarabine.

5. Use of a compound of formula (II) as claimed in any one of claims 1 to 4 in the manufacture of a medicament for the treatment of leukemia in a mammal.

6. Use according to claim 5, wherein the leukemia is selected from acute myelocytic leukemia, para-acute monocytic leukemia, acute lymphocytic leukemia.